Matrix Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry (MALDI-IMS) can support modern pathology by precisely identifying “biomarker” molecules, whose identity and location in a tissue sample indicate the existence and progression of a specific disease (Caprioli et al., 2008). However, three primary problems intervene. One such problem involves resolving isobaric ions which have the same mass-to-charge ratio (m/z), but different structures. This problem prevents hundreds of important biomolecules weighing less than 2 kDa from being uniquely identified by mass spectrometry alone. Another problem associated with analyzing biomarker molecules through MALDI-IMS is that tens of thousands of matrix molecules are required to desorb and ionize one molecule of bio-analyte which causes the applied matrix films to be thicker than the tissue slice to be analyzed (Dreisewerd, 2003). Another typical problem is that neutral molecules are desorbed almost exclusively which means that very few ions are directly produced by MALDI for analysis. Ultimately, these problems contribute to the practical mass spectrometry imaging signal being limited as the laser spot size decreases.
For example, the intracellular analysis of single cells by MALDI is not possible because of the aforementioned problems. In fact, presently, practical molecular analysis by MALDI of any surface smaller than 100 square microns is very difficult because (1) an excess of matrix is required to activate the available analyte, (2) overlapping spectral interferences are difficult to interpret by mass spectrometry alone, and (3) poor ionization efficiency limits sensitivity. All of these factors limit the analysis of a small surface to only the identification of the easily ionizable majority molecular components on the cell or tissue surface. Despite these problems, remarkable progress has been made in applying MALDI-IMS to real world issues (Caprioli et al., 2008). Moreover, unique instrumentation and analytical procedures have begun to appear over the last ten years to separately address each of the limitations of MALDI-IMS (Sinha et al., 2007).
Recently, combinations of either pulsed electrospray or MALDI with IM-oTOFMS have revived the use of ion mobility for bioanalysis by not only providing separation of conformers, but separation based on charge state as well. In the case of MALDI, the useful mass range can simultaneously encompass from low mass elements to 300 kDa mass biocomplexes.
In general, ion mobility (IM) is used to separate gas phase ions by forcing the ions to traverse an electrically biased cell filled with an inert gas such as helium. The electrical acceleration of an ion in the ion mobility cell is restricted by many low energy collisions with the helium atoms such that the average drift velocity with which that ion moves is proportional to its shape. For example, a molecule with sixty carbon atoms moves nearly twice as fast when it is in the spherical form of a “buckyball” compared to a nearly flat graphene sheet (Von Helden et al., 1993; and, Shvartsburg et al., 1999). Ion mobility became an extremely potent tool for sorting nearly isobaric gas phase cluster ion structures. This was first realized when it was first shown that the combination of a pulsed ion source at the entrance of the ion-mobility cell entrance and an orthogonal time of flight analyzer at the cell exit could uniquely determine both mass-to-charge ratio and ion mobility drift time for each and every ion from a sample.
The notoriously difficult MALDI analysis of small molecules in tissue has been tremendously assisted by MALDI-ion mobility orthogonal time-of-flight mass spectrometry, in particular, MALDI-IM-oTOFMS (Jackson et al., 2007). “Chemical noise” is the euphemism for the unavoidable, unresolved ion signal which hinders or prevents the interpretation of MALDI spectra at a mass-to-charge ratio of less than about 1000 Da. When MALDI-IM-oTOFMS is applied, this otherwise worthless spectral background is separated into useful familial trend lines rich with conformational information that becomes clear in displays of ion mobility drift time versus mass-to-charge ratio. These trend lines uniquely identify the presence of lipids, peptides, nucleotides, and small molecules (including matrix ions) in tissue.
As described herein, the present disclosure provides a method and apparatus for ionizing the largely ignored neutral MALDI desorption plume, and in particular, for efficiently measuring the ionized MALDI desorption plume when post-ionization (POSTI) techniques are combined with a medium pressure MALDI-Ion mobility orthogonal time-of-flight mass spectrometry (MALDI-IM-oTOFMS) instrument. Additionally, the present disclosure provides a method and apparatus that simultaneously separates tissue-sample MALDI ions by IM-oTOFMS according to their chemical family, and then directly compares these MALDI ions to the ions created by post-ionizing the co-desorbed neutral molecules with a second laser which is time-delayed, typically by a few hundred microseconds. Also, the present disclosure provides a method and apparatus for using post-ionization to identify intact molecules of cholesterol, lipids, peptides, proteins, and giant fullerenes that may be present on tissue surfaces, spatial imaging of post-ionized molecules (e.g. cholesterol) in brain tissue, and indentifying controllable photo-fragmentation for in-situ identification of proteins and peptides. The present disclosure further provides novel approaches that enhance the analysis of ions, including the use of giant fullerene internal standards to enhance mass accuracy, and ultraviolet (UV) declustering lasers to generate intact peptides and proteins, followed by vacuum ultraviolet (VUV) post-ionization which generates identifying structural fragments.